FRACTIONATION OF FICUS DELTOIDEA LEAVES EXTRACT USING SOLID

PHASE EXTRACTION ON ANTI-AGEING ACTIVITY IN VITRO

FATIN HUSNA BINTI ZULIKRAM

A thesis submitted in fulfilment of the

requirements for the aw ard of the degree of

Master of Philosophy

School of Chemical and Energy Engineering

Faculty of Engineering

Universiti Teknologi Malaysia

APRIL 2019

V

ABSTRACT

Ficus deltoidea (F. deltoidea) belongs to moraceae. Vitexin and isovitexin

that have high antioxidant property are considered as the chemical markers of F.

deltoidea leaves. The objective of this research was to investigate the fractionation of

F. deltoidea leaves using solid phase extraction on anti-ageing activity in vitro. The

leaves of F. deltoidea were extracted by methanol by using ultrasonic extraction

method and further fractionated using different concentration of methanol.

Antioxidant activity of F. deltoidea leaves fractions and extract was analyzed

through 2,2-diphenyl-1-picrylhrazyl (DPPH) scavenging ability test and total

flavanoid content (TFC) activity. The result shows that 80% methanol 20% water

fraction (F4) have higher percentage of scavenging activity and flavonoid content

with 87.16% and 237.57 mg, respectively. The quantification of biomakers (vitexin

and isovitexin) was performed using high performance liquid chromatography

(HPLC) and the result showed that F4 fraction have higher percentage of vitexin

(11.02%) and isovitexin (0.49%) compared to other fractions and extract. Moreover,

cytotoxicity study on human skin Fibroblasts cell (HSF 1184) also demonstrated that

F4 has higher percentage of cell viability with value 175.29% at 100 jig/mL. The

anti-ageing activity of F. deltoidea leaves fractions and extract were further

evaluated using sircol collagen assay, inhibition of elastase assay, hyaluronidase

assay and lipoxygenase assay, where F4 also showed the strongest activities

compared to other fractions and extract for all assays with the value of 8.9 jig

collagen concentration, 83%, 97.82%, and 88.56%, respectively. Taken together, as

F4 contains high amount of vitexin and isovitexin, therefore these compounds have

the potential to be further developed as anti-ageing agent.

ABSTRAK

Ficus deltoidea (F. deltoidea) tergolong dalam moraceae. Viteksin dan

isoviteksin yang mempunyai antioksida yang tinggi merupakan penanda kimia yang

terdapat dalam daun F. deltoidea. Objektif kajian ini dilakukan untuk pemeringkatan

daun F. deltoidea menggunakan pengekstrakan fasa pepejal secara in vitro untuk

aktiviti anti-penuaan. Daun F. deltoidea diekstrak menggunakan metanol dan

pemeringkatan dijalankan menggunakan kepekatan metanol dengan kaedah

ultrasonik yang berbeza. Aktiviti antioksida pecahan dan daun mentah F. deltoidea

dianalisis melalui ujian aktiviti pemerangkapan 2 ,2-diphenil-l-picrilhrazil dan

jumlah kandungan flavanoid. Keputusan yang diperolehi menunjukkan eampuran

80% metanol : 20% air (F4) mempunyai peratusan yang tertinggi bagi aktiviti

memerangkap dan kandungan flavanoid masing-masing dengan nilai 87.16% dan

237.57 mg. Pengukuran biomarker (viteksin dan isoviteksin) ditentukan

menggunakan kromatografi cecair prestasi tinggi dan keputusan menunjukkan

pecahan F4 mempunyai peratusan viteksin (11.02%) dan isoviteksin (0.49%) yang

tinggi berbanding ekstak dan pecahan yang lain. Tambahan pula, kajian sitotoksik

terhadap kulit manusia dijalankan menggunakan sel fibroblast (HSF 1184)

menunjukkan F4 juga mempunyai peratusan daya maju sel tertinggi iaitu 175.29%

pada kepekatan 100 pg/mL. Kajian aktiviti anti-penuaan terhadap pecahan dan

ekstrak daun F. deltoidea seterusnya dikaji melalui kaedah kolagen sirkol,

perencatan elastase, hialuronidase dan liposiginase mendapati F4 juga menunjukkan

aktiviti yang terkuat berbanding pecahan dan ekstrak keatas kesemua aktiviti

masing-masing dengan nilai 8.9 pg , 97.82%, 97.82% dan 88.56%. Secara

keseluruhannya, F4 mempunyai kandugan viteksin dan isovteksin yang tinggi di

mana sebatian ini berpotensi dibangunkan sebagai agen anti-penuaan.

ACKNOWLEDGEMENT

First of all, praise to Allah for giving me strength and good health in

completing my research project.

1 would like the express my sincere gratitude to my supervisors, Dr Rosnani

Hasham for her constant guidance, kind assistance and precious time for discussion

throughout of this research project. I would like to thank all the lab mates for their

support and encouragement.

Besides, I would like to record my special thanks to my lovely husband

Mohammad Khaidree for his support physically, mentally and financially and

encouragement in completing this research. I would like to extend my thanks to my

parents Zulikram Mustapa and Roselina Mustaffa for their encouragement, love and

advice throughout in completing this research project. Not to forget also, to my two

cutic babies Luqman Hakim and Hanna Humaira for behaving well and let your

mummy completing her master research.

TABLE OF CONTENTS

CHAPTER TITLE PAGE

ACKNOWLEDGEMENT

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENT vii

LIST OF TABLES viii

LIST OF FIGURES ix

LIST OF ABBREVIATIONS x

1 INTRODUCTION 1

1.1 Research Background 1

1.2 Problem Statement 4

1.3 Objective 5

1.4 Scope of Research 6

1.5 Significant of study 6

2 LITERATURE REVIEW 7

2.1 Skin 7

2.1.1 Elements in the skin 8

2.1.1.1 Skin Cells 8

2.1.1.2 Skin Fibers 9

2.2 Ageing 10

2.3 Skin Ageing 10

2.3.1 UV radiation and its effects on the humanskin 11

2.3.2 Mechanisms of skin ageing 13

2.3.3 Hyaluronic acid and skin ageing 14

2.4 Ficus deltoidea 16

2.4.1 Phytochemical properties of Ficus deltoidea 17

2.4.1.1 Vitexin 18

2.4.1.2 Isovitexin 19

2.4.1.3 Chemical features of vitexin andisovitexin 19

2.4.2 Biological activity of Ficus deltoidea 21

2.4.2.1 Anti-inflammatory activity 21

2.4.2.2 Antioxidant activity 22

2.5 Ultrasonic Assisted Extraction 23

2.6 Solid Phase Extraction 25

2.6.1 The Effect of Fractionation ofPhytochemical on Health Benefits 28

3 METHODOLOGY 30

3.1 Introduction 30

3.2 Chemicals 32

3.3 Preparation of Raw Materials and ExtractionProcess ^3

3.4 Fractionation of Ficus deltoidea extract usingSolid Phase Extraction 33

3.5 Quantification of vitexin and isovitexin in Ficusdeltoidea leaves fractions 35

3.5.1 High Performance Liquid Chromatography(HPLC) 35

3.6 Analysis on antioxidant activity 36

3.6.1 DPPH radical scavenging activity 36

3.6.2 Total Flavanoid Content (TFC) 36

3.7 Effect of Ficus deltoidea leaves extract andfractions on anti-ageing activity 3-7

3.7.1 Cell Line 37

3.7.2 Cell Seeding 37

3.7.3 Medium Renewal 37

3.7.4 Cell counting and evaluation on viable cells 38

3.7.5 Cell viability assay 38

3.7.5.1 UV irradiation and treatment 38

3.7.5.2 MTT assay 39

3.8 Measurement of Collagen content by SircolCollagen Assay (SCA) 39

3.8.1 Sample Preparation 40

3.8.2 Determination of Collagen Assay 40

3.9 In vitro elastase inhibition assay 41

3.9.1 Preparation of Tris-HCL buffer 41

3.9.2 Preparation of enzyme solution 41

3.9.3 Preparation of substrates 41

3.9.4 Serial dilution of Ascorbic Acid 41

3.9.5 Serial dilution of crude extract and fractions 42

3.9.6 Determination of elastase inhibition activity 42

3.10 Anti-inflammatory activity of Ficus deltoidealeaves fraction 43

3.10.1 Hyaluronidase inhibitory assay 43

3.10.2 Lipoxygenase inhibitory assay 44

3.11 Statistical analysis 45

4 RESULTS AND DISCUSSION 46

4.1 Detection and Quantification of vitexin andisovitexin in Ficus deltoidea leaves fraction 46

4.2 Antioxidant activity of Ficus deltoidea leavesfractions 4g

4.3 Anti-ageing Activity of Ficus deltoidea 52

4.3.1 Cell Cytotoxicity Study 52

4.3.2 Collagen synthesis study 55

4.3.3 Elastase Assay 57

4.3.4 Anti-inflammatory activity of F. deltoidealeaves fractions 59

5 CONCLUSION 62

REFRENCES 63

LIST OF TABLE

TABLE NO. TITLE PAGE

2.1 The description of skin cells 9

2.2 Description of skin fibers 9

2.3 The structure of compound isolated from F. deltoidea 18

2.4 Type of extraction method 23

3 .1 List of chemical and solvent used 32

3.2 Sample ritual of for each well in 96-well plate for elastaseinhibition assay of F. deltoidea leaf fraction and crude ^extract.

4.1 Quantitative analysis of vitexin and isovitexin in Ficusdeltoidea leaves fractions 47

4.2 IC50 of Inhibition of elastase assay by F. deltoidea leaves ^fractions.

LIST OF FIGURE

FIGURE NO TITLE PAGE

2.1 Structure of human skin 7

2.2 UVA and UVB penetration into human skin 11

2.3 Mechanisms of ageing 13

2.4 Photo of Ficus deltoidea 16

2.5 Structure of vitexin and isovitexin 20

2.6 Schematic diagram of ultrasound-assisted extraction 24

2.7 SPE method 27

3.1 Project outline 30

3.2 An illustration of SPE fractionation process 33

3.3 Block of diagram HPLC system 34

3.4 Sircol collagen assay flowchart 39

4.1 The percentage of free radical scavenging activityobserved among fractions. 48

4.2 Percentage of total flavanoid content in F. deltoidea 50

4.3 Cell viability of HSF cells cultured with increasing concentration of F. deltoidea leaves fractions and extractusing MTT assay 53

4.4 Collagen contains in F. deltoidea leaves fractions 55

4.5 F. deltoidea leaves fractions on inhibition of elastaseassay 56

4.6 Inhibition of Hyaluronidase assay activity of F.s deltoidealeaves fractions and extract 59

4.7 Inhibition of Lipoxygenase assay activity of F. deltoidealeaves fractions and extract 60

LIST OF ABBREVIATION

UV Ultra violet

ROS Reactive Oxygen Species

AP-1 Activator protein-1

MMP Matrix mctalloprotcinasc

ECM Extracellular matrx

SPE Solid phase extraction

MMP-1 Matrix metalloproteinase-1

MMP-9 Matrix metalloproteinase-9

MMP-3 Matrix metalloproteinase-3

HA Hyaluronic acid

GAG Glycosaminoglycan

HYAL 1 Iyaluronoglucosaminidasc

DFT Density Functional theory

iNOS inducible nitric oxide synthase

DPPH 2,2-Diphenyl-1 -picrylhydrazy 1

UAE Ulrasound-assisted extraction

TFC Total flavonoid content

MTT 3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide

LOX Lipoxygenase

HPLC High performance liquid chromatography

UPM Universiti Putra Malaysia

MeOH Methanol

FI Fraction 1

F2 Fraction 2

F3 Fraction 3

F3 Fraction3

F4 Fraction4

F5 Fraction5

TFA Trifloroaceticacid

DM EM Dulbecco’s modified essential medium

FBS Fetal Bovine Serum

C 02 Carbon dioxide

HSF Human skin fibroblast

DMSO Dimethyl sulfoxide

SCA Sircol collagen assay

HCL Hydrochloric acid

SANA 5 mM N-Succinyl-Ala-Ala-Ala-nitroanilide

HNE human neutrophil elastase

Na2H2P 0 4 Sodium phosphate

NDGA Nordihydroguaiaretic acid

SPSS Statistical Package Social Scicncc

ANOVA Analysis of variance

UAE Ultrasonic assisted extraction

CHAPTER 1

INTRODUCTION

1.1 Background of Study

Ageing is a natural phenomenon, a physiological change that is certain to be

experienced by each living organism. It is a complex metabolic process that occurs

over a period of time through growth, development and maturity. The ageing process

is said to happen when the energy to maintain the structural and functional molecules

that are synthesized in our life is no longer accessible (Hayflick, 2004). As the bodies

reach maturity, the skin appearance and characteristics change. In general, ageing is

more prominently seen on skin where thin, dry, unblemished, elasticity-depleted

texture and fine wrinkles are the common indicators (Rogers et al., 2008).

Skin is the largest organ of the body with multiple cell types and structures

that exhibits multiple functions, among them is a protcctivc barrier between internal

organs and outer environment (Feamley, 2009). The outer part of skin is composed

of fibro, protein, collagen and elastin. Collagen is one of the major building block of

skin which responsible for elasticity and strength of the skin.

Skin ageing can be divided into intrinsic and extrinsic ageing (Bennett &

Cooper, 2009). Intrinsic ageing is determined primarily by oxidative metabolism,

2

genetic and hormonal factors while extrinsic ageing emphasize on the exposure to

the solar ultraviolet (IJV) radiation. This IJV radiation will induce free radical

damage and increase Reactive Oxygen Species (ROS), which responsible for

oxidative stresses and inflammatory responses in the dermal or epidermal layer by

destructing the connective tissues fibers. Free radical damage will bring about DNA

damage, protein and gene modifications. High level of ROS will induce the

transcription of Activator protein 1 (AP-1). AP-1 is responsible in regulation of cell

growth and differentiation. AP-1 strongly regulated the transcription of Matrix

metalloproteinase (MMP), which causes further degradation of mature fibrillar

collagen in the skin which contributes to skin ageing (Yin, 2014). Bissett et al (1987)

reported that the decrease of skin elasticity with premature ageing is significantly

correlated with increased elastase and hyaluronidase activity. Therefore, the

inhibition of these enzymes may be the most effective therapy to improve the

structure of collagen in the extracellular matrix (ECM) and control its metabolism

(Mukherjee et al., 2011).

The widespread awareness on depletion of ozone layer and the danger of UV

radiation reaching directly to the surfacc of the Earth has made the socicty at large

become more alert regarding the effect of this harmful ray on their skin.

Nevertheless, skin care is not just a matter of health but an affair of beauty as w7ell,

which lead to the ever growing skin-based research conducted by various interested

parties. In the turn of the decade, high accessibility of information regarding skin

care products by consumer demands a greater need for anti-ageing products that are

scientifically-proven in its efficiency (Rogers et al., 2008).

Consuming proper food choice might be the larger part of what makes skin

age gracefully while remaining healthy, strong and disease-free. Powerful anti­

ageing benefits can be obtained from wide range of natural food, spices and herbs

that carry high incidence of antioxidant properties. Traditional herbs have been

proved to be safe and effective for ageing-related problem and very much often

attracted the growing industry of skin-care product with niche in herb-based

medicine (Hoffmann, 2013). Treatment of the skin with products containing plant-

3

derived anti oxidant ingredients has been proven useful for the prevention of UV-

mediated cutaneous damage. Various parts of the plant are available to be processed

and consumed in the form of powder, tablets and extract.

This study attempts to focus on Ficus deltoidea, (F deltoidea) a traditional

herb from the genus Ficus of Moraceae family. F. deltoidea, locally known as ‘Mas

Cotek’ is widely distributed in Peninsular Malaysia and a popular medicinal herb

among Malay. This miraculous herb has been used traditionally to treat wounds and

sore, used as an antidiabetic treatment and an after-birth tonic to contract the uterus

and vaginal muscle (Bunawan et al., 2014). F deltoidea also reported to possess

powerful antioxidant activity which is good for anti-ageing (Mohd el al., 2015).

Bunawan et al. (2014) reported the biological properties of F deltoidea

which are antioxidant, antidiabetic, anti-inflammatory, anti-ulcerogenic, wound

healing activity, anti-bacterial activity and anticancer activity. Wide ranges of

chemical compound have been identified from leaves of F deltoidea. Some of the

compound that is stated to be present in the F deltoidea is flavonoids such as

isovitexin, vitexin, proanthocynidins, flavan-3-ol monomers and flavones glycosides

(Misbah et al., 2013). The volatile compound identified is mainly product of

shikimic acid pathway, terpenoids and aliphatic groups. Vitexin and isovitexin has

been reported can be effective for the prevention of free radical scavenging (Kim et

al., 2005). Isolation and purification process are important to identify the bioactive

compound in F deltoidea leaves.

Fractionation is a separation process in which a certain quantity of a mixture

is divided into smaller quantities in which of the composition vary’ according to the

gradient (Baynes, 2017). The use of fractionation could isolate the structural and

morphology of identifiable entity for subsequent analysis, the emphasis being on

purity at the expense of yield. During Solid phase extraction (SPE), the target analyte

and structurally related compounds arc adsorbed onto a solid, stationary phase. The

solid phase is then washed with selective eluents in order to eliminate any interfering

4

substances and to reduce the complexity of the matrix. Finally, the bound target

analyte is recovered by an elution step (Sigma, 1998). The solvent should enable

rapid elution of the analyte from the solid phase. The recovery of organic compounds

by SPE is highly dependent on the polarity of the eluents. A study conducted by

Barbosa et al (2013) proved that purification obtain from the fractionation process

yield higher antioxidant activity than the crude extract. Therefore, by applying this

approach, reliable information about the potential of chemical compound in the F.

deltoidea extracts on anti-ageing activity could be well understood and ultimately, a

product which can slow the ageing process and make for a younger-looking skin

could be produced.

1.2 Problem Statement

Exposure of the skin to UV occurs from both natural and artificial sources.

The sun is the main source of UV radiation on human while the depletion of the

ozone layer would intensifies the harmful exposure to humankind and the

environment. As stated by Sudcl et al. (2005), people without natural protection arc

estimated to account up to 90% visible skin ageing due to the effect of sunlight on

the skin. As people age, their concern tow'ards outward appearance increases

profoundly and the concept of delaying the process of ageing seems appealing to

most person. Today’s anti-ageing market is expanding to incorporate diverse

consumer concerns.

Recently, many herbs and natural products has been receiving public interest

as complement and alternative medicine. Herbs which contain antioxidant and anti­

inflammatory are used in cosmetic and dermatological products to improve signs of

extrinsic ageing (Ho et al., 2010). F. deltoidea extracts have been found to have

photo-protective effects on epidermal cell line. A study reported by Mohd et al.

(2015) stated that F. deltoidea has very strong antioxidant properties. Antioxidant

can neutralize and stimulate the production of collagen and restore skin elasticity,

thus can slow down the ageing process (Watson, 2013). Moreover, Zino et al. (1997)

5

reported the ability of F. deltoidea antioxidant that can delay some effects o f ageing.

A study conducted by Hasham et al. (2011) reported that skin ageing was strongly

related to the inflammatory process. Leaves of F. deltoidea also have been confirmed

devoid any toxic elements as reported by Shafei et al. (2011) and Farsi et al. (2013)

also showed that F. deltoidea do not have any potential to induce mutation.

Flavanoids are a class of secondary plant phenolics with significant

antioxidant and chelating properties (Seawan & Jimtaisong, 2013). Furthermore,

flavonoids protect plants from solar UV radiation and scavenge UV generated ROS

(Shirley, 1996). Therefore, flavonoids have three different photoprotcction effects

including UV absorption, direct and indirect antioxidant properties, and modulating

several signaling pathways. Vitexin and isovitexin are flavoncs, which is one kind of

flavanoid. These compound have been drawing more attention antioxidant activity

and anti-inflammatory activity that can help in slowing the ageing process (He et al.,

2016). In order to isolate the bioactive compound in F. deltoidea leaves, fractionation

method by using Solid Phase Extraction (SPE) was used in this study. Therefore, the

proposed project is expected to yield novel insight on F. deltoidea leaves fractions on

anti-agcing cffccts and lead to better understanding of anti-agcing properties of F.

deltoidea leaves which is important and can give benefits to pharmaceutical and

cosmeceutical industry.

13 Objective

The objective of this research was to investigate the fractionation of Ficus

deltoidea leaves using solid phase extraction on anti-ageing activity in vitro.

6

1.4 Scope of Research

The scopes of research were:

1. Fractionation of F. deltoidea leaves extract by using solid phase extraction

(SPE) method.

2. Determination of antioxidant properties of F. deltoidea leaves fractions using

DPPH free radicals scavenging assay and total flavanoid contcnt.

3. Evaluation of vitexin and isovitexin in F. deltoidea leaves fractions using

HPLC.

4. Observation and investigation of anti-ageing effects caused by UVB

irradiation of F. deltoidea leaves fractions on fibroblast ccll line.

5. Evaluation of anti-ageing properties of F. deltoidea leaves fractions by using

sircol collagen assay, MTT assay, elastase assay, hyaluronidasc assay, and

lipoxygenase assay.

1.5 Significant of Study

The finding of this study will rebound the benefit to pharmaceutical and

cosmeceutical industry. From the result, fractionation of bioactive compound by

using SPE method can isolate vitexin and isovitexin at mixture of 80% methanol :

20% w'ater. This fraction shows the highest ability to slow down the ageing problem

by ability to proliferate high percentage of fibroblast cells, produce more collagen

contcnt, inhibit elastase activity lipoxygenase and hyaluronidasc assay.

63

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